Attenuated enterohemorrhagic E. coli-based vaccine vector and methods relating thereto

ABSTRACT

An attenuated enterohemorrhagic  E. coli -based vaccine vector is disclosed. Enterotoxigenic  E. coli  colonization factor antigen 1 and the B subunit of  E. coli  heat labile toxin have been expressed in the attenuated enterohemorrhagic  E. coli  vector strain. Immunized animals are further protected against lethal and non lethal challenges with the enterotoxigenic  E. coli  strain. Immunization of mice with the vaccine construct induces mucosal antibody against both antigens, establishing the attenuated  E. coli  vector strain as a generally useful vector for presenting one or more antigens to a subject in a vaccine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the §371 U.S. National Stage of InternationalApplication No. PCT/US2010/040522, filed 30 Jun. 2010, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/221,612,filed Jun. 30, 2009, each of which is hereby incorporated by referencein its entirety.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.1R21AI079711-01A1, awarded by the National Institutes for Health,National Institutes of Allergy and Infectious Disease. The Governmenthas certain rights in this invention.

BACKGROUND

Enterotoxigenic E. coli (ETEC) are important bacterial pathogens causingworldwide morbidity and mortality. Enterotoxigenic E. coli infectionsare important causes of death in infants and children under the age offive in developing countries. Illness caused by an enterotoxigenic E.coli infection is often self-limiting, lasting about one week. However,the illness can range from a mild diarrhea with little to no dehydrationto a very severe and potentially fatal cholera-like disease,particularly in infants. Enterotoxigenic E. coli are also the leadingcause of diarrhea in travelers to high-risk areas.

Despite our good understanding of ETEC virulence factors, and althoughseveral potential ETEC vaccines tested in volunteer trials and fieldstudies, no safe and effective vaccine is yet available for at-riskindividuals. Safe and effective ETEC vaccines would have a considerablepublic health impact worldwide in infants in developing countries, intravelers from industrialized countries to the developing world, and forthe military.

SUMMARY OF THE INVENTION

The present invention relates to methods that involve administering acomposition to a subject in order to induce the subject to generate animmune response against one or more components of the composition.Generally, the method includes administering to a subject a compositionthat includes an attenuated enterohemorrhagic E. coli (EHEC) in anamount effective to induce the subject to generate an immune responseagainst at least one immunogen expressed by the attenuated EHEC.

In some embodiments, the immunogen can be an immunogen naturallyexpressed by the EHEC. In other embodiments, the immunogen can be aheterologous immunogen such as, for example, an immunogen that isexpressed by the attenuated EHEC from a heterologous polynucleotide thatencodes the heterologous immunogen. Thus, in some embodiments, theattenuated EHEC can include a heterologous polynucleotide that encodesone or more heterologous immunogens.

In some embodiments, the wild-type of the attenuated EHEC can a pathogento the subject.

In some embodiments, administering the composition to the subject canprotect the subject against challenge by an enterotoxigenic E. coli.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows properties of wild-type ETEC, ZCR533 EHEC vector,ZCR533-CFA/I vaccine construct, and ZCR533-CFA/I+mLT vaccine construct.¹TEM with Negative staining (0.25% Phosphotungstic acid) reveals CFA/Ifimbriae surrounding the ZCR533(pGA-CFA/I) andZCR533(pGA-CFA/I-truLThK63) vaccine strains as well as the ETEC H10407wild-type strain. No fimbriae were seen on the vector. ²TheCFA/I-expressing vaccine strains and wild-type H10407 strain inducecharacteristic mannose resistant agglutination of type A red blood cellsnot seen with ZCR53 alone. ³The characteristic hydrophobicity of CFA/Iprotein (believed to aid in overcoming repulsive electrostatic forcesduring adherence) was seen in WT H10407 and with the EHEC CFA/I and EHECCFA/I-mLT constructs. Expressing CFA/I, which aggregated at low saltconcentrations [0.08M]. The ZCR522 vector, which has hydrophilic surfaceproperties only aggregated at a high salt concentration [4 M]. ⁴Only theZCR533(pGA-CFA/I-truLThK63) vaccine and the ETEC H10407 wild-typestrains were positive for LT production, whereas, the ZCR533 vector andZCR533(pGA-CFA/I) vaccine non-LT producing strains were negative in thisassay. ⁵In vitro stability of the plasmids containing a kanamycinresistance marker were shown by serial passage of the strains in brothfor ten 12 hour periods (100 generations) followed by plating of thestrains on agar with and without kanamycin. ⁶In vivo stability of theplasmids was shown following intranasal administration of the vaccinesto mice by culturing lung contents daily for ten days and quantitatingthe numbers of kanamycin-resistant CFUs.

FIG. 2 shows serum α-CFA/I IgG response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 3 shows serum α-CFA/I IgM response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 4 shows serum α-CFA/I IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 5 shows serum α-LT IgG response in mice vaccinated with ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 6 shows serum α-LT IgM response in mice vaccinated with ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 7 shows serum α-LT IgA response in mice vaccinated with ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 8 shows serum α-Intimin IgG response in mice vaccinated with ZCR533EHEC 7.5 vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 9 shows serum α-Intimin IgM response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 10 shows serum Intimin IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 11 shows serum α-O157 LPS IgG response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 12 shows serum α-O157 LPS IgM response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 13 shows serum α-O157 LPS IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 14 shows nasal α-CFA/I IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 15 shows lung α-CFA/I IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 16 shows small intestine α-CFA/I IgA response in mice vaccinatedwith ZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 17 shows fecal pellet α-CFA/I IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 18 shows nasal α-LT IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 19 shows lung α-LT IgA response in mice vaccinated with ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 20 shows small intestines α-LT IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 21 shows fecal pellet α-LT IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 22 shows nasal α-Intimin IgA response in mice vaccinated withZCR533 EHEC 7.5 vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 23 shows lung α-Intimin IgA response in mice vaccinated with ZCR533EHEC vector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 24 shows small intestines α-Intimin IgA response in mice vaccinatedwith ZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 25 shows fecal pellet α-Intimin IgA response in mice vaccinatedwith ZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 26 shows nasal α-O157 LPS IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 27 shows lung α-O157 LPS IgA response in mice vaccinated withZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 28 shows small intestines α-O157 LPS IgA response in micevaccinated with ZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct,or the ZCR533-CFA/I+mLT vaccine construct.

FIG. 29 shows fecal pellet α-O157 LPS IgA response in mice vaccinatedwith ZCR533 EHEC vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct.

FIG. 30 shows inhibition of wild-type ETEC H10407 binding to Caco-2cells by mouse antiserum generated against the ZCR533 EHEC vector, theZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLT vaccineconstruct.

FIG. 31 shows inhibition of LT-mediated elongation of CHO cells by mouseantisera raised against the ZCR533 EHEC vector, the ZCR533-CFA/I vaccineconstruct, or the ZCR533-CFA/I+mLT vaccine construct.

FIG. 32 shows inhibition of LT-binding to GM1 by mouse anti-serumagainst ZCR533 vector, ZCR533-CFA/I vaccine, or ZCR533-CFA/I+mLT vaccinestrains.

FIG. 33 shows inhibition of LT-B-binding to GM1 by mouse anti-serumagainst ZCR533 vector, ZCR533-CFA/I vaccine, or ZCR533-CFA/I+mLT vaccinestrains.

FIG. 34 shows clearance of intranasally administered wild-type ETECH10407 from mouse lung after vaccination with the ZCR533 EHEC vector,the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLT vaccineconstruct. *P<0.05

FIG. 35 shows inhibition of wild-type ETEC H10407 colonization of thesmall intestine after intranasal vaccination with the ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 36 shows inhibition of wild-type ETEC H10407 colonization of thesmall intestine after intragastric vaccination with the ZCR533 EHECvector, the ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct.

FIG. 37 shows inhibition of intestinal fluid accumulation afterintragastric vaccination with the ZCR533 EHEC vector, the ZCR533-CFA/Ivaccine construct, or the ZCR533-CFA/I+mLT vaccine construct, thenintragastric administration of heat-labile enterotoxin (25 μg).

FIG. 38 shows inhibition of intestinal fluid accumulation afterintragastric vaccination with the ZCR533 EHEC vector, the ZCR533-CFA/Ivaccine construct, or the ZCR533-CFA/I+mLT vaccine construct, thenadministration of wild-type ETEC H10407.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

We have developed live, attenuated vaccine vectors based onenterohemorrhagic E. coli (EHEC) strains. In some cases, the vector ismodified to express one or more immunogens such as, for example, one ormore protective enterotoxigenic E. coli antigens. As our vaccine vector,we use an attenuated attaching/effacing E. coli strain with minimalreactogenicity derived from an O157:H7 enterohemorrhagic E. coliisolate. We have prepared attenuated, live vaccine constructs byintroducing plasmids containing cloned segments of DNA encodingrepresentative immunogens into the attenuated EHEC vector. Asrepresentative immunogens, we have selected certain virulencedeterminants of enterotoxigenic E. coli (e.g., the colonization factorantigen I (CFA/I) and the heat-labile enterotoxin (LT)). To test thesafety, immunogenicity, and protective efficacy of the constructs we usea novel mouse model of intranasal immunization and enterotoxigenic E.coli challenge. The attenuated, live vector, modified to expressenterotoxigenic E. coli virulence determinants induces a protectiveimmune response against a future lethal enterotoxigenic E. colichallenge. These studies have resulted in the development of a safe andeffective live, attenuated vector. One use for such a vector is theconstruction and administration of a vector-based vaccine directedagainst enterotoxigenic E. coli infections.

The term “immunogen” and variants thereof refer to any material capableof inducing an immune response in a subject challenged with thematerial. An immunogen may occur naturally or reflect a truncated,chimeric, or otherwise modified version of a naturally occurringimmunogen. Thus, an immunogen may represent a full length protein orpolypeptide as naturally expressed or may represent an immunogenicportion or may reflect a modified version of such a polypeptide havingat least one addition, deletion, or substitution.

The term “protect” and variants thereof refers to any decrease in thelikelihood and/or extent of infection and/or pathology due to infection,including, for example, any amelioration and/or decrease in theoccurrence and/or severity of clinical signs or symptoms of a conditioncaused by infection by the pathogen.

The term “sign” or “clinical sign” refers to an objective physicalfinding relating to a particular condition capable of being found by oneother than the patient.

The term “symptom” refers to any subjective evidence of disease or of apatient's condition.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

Enterotoxigenic E. coli are noninvasive and colonize the small intestineby attachment to mucosal receptors via surface-expressed hair-likefimbrial colonization factor adhesins (e.g., CFA/I, CS1, CS2, CS4, CS5)or finer fibrillar colonization factor adhesins (e.g., CS3, CS6) made ofrepeating structural subunits. The binding of enterotoxigenic E. coli tospecific glycoprotein receptors or glycolipid receptors present on themucosa is mediated by a subset of adhesive subunits that may beexpressed at the tips of fimbriae. Intact fimbriae, or their structuralor adhesive subunits, are appropriate targets for inclusion in vaccinesin order to induce mucosal antibodies that will prevent small bowelcolonization. Certain colonization factor antigens associated withenterotoxigenic E. coli strains have been grouped into families that mayshare immune determinants. Three colonization factors (CFA/I, CS3, andCS6) are expressed by the majority of enterotoxigenic E. coli isolates.

Enterotoxigenic E. coli that adhere to the mucosa of the small intestineproduce heat-labile (LT) and/or heat-stable (ST) enterotoxins.Expression of these toxins can result in a watery, non-inflammatorydiarrheal disease in the host. The primary cause of morbidity andmortality is dehydration.

Heat-labile toxin (LT) is a typical A1B5 toxin whose pentameric Bsubunit binds to its GM1 ganglioside host receptor, allowing the Asubunit to activate adenylate cyclase resulting in increased cyclic AMPand enhanced chloride secretion. LT components are appropriate forinclusion in vaccines to target bacterial strains that express only LTas well as strains that express both LT and heat-stabile (ST)enterotoxins. The B subunit of LT is an effective immunogen andprotective antigen, whereas the holotoxin has important immune adjuvantproperties. Mutants of the holotoxin have been developed which have lostsecretory activity but maintain adjuvant activity.

Heat-stable toxin (ST) is a small molecule that binds guanylate cyclasethereby increasing cyclic GMP, net chloride secretion, and, therefore,watery diarrhea. ST may not induce immunity in natural infection. SinceST has not been shown to be a reliable, practical vaccine target,immunity to strains that express ST but not LT typically exploit theimmunogenicity of other antigens such as, for example, adhesins.

More than one hundred serotypes associated with enterotoxigenic E. colibased on lipopolysaccharide (O) and flagellar (H) typing have beenidentified. Common serotypes account for only about 15% of theenterotoxigenic E. coli in individual studies. Therefore,lipopolysaccharide and/or flagellar antigens are less suitable targetsfor designing broad spectrum enterotoxigenic E. coli vaccines.

At present, there is no licensed enterotoxigenic E. coli vaccine.Enterotoxigenic E. coli vaccine development is geared towards inducingmucosal immune responses to (a) block initial colonization ofenterotoxigenic E. coli in the gut and (b) neutralize enterotoxigenic E.coli toxins. Epidemiological studies, as well as volunteer trials andanimal studies, have shown that acquired immunity to enterotoxigenic E.coli infection is mainly a result of anti-colonization factor immuneresponses. Thus, mucosal antibodies against CFAs can induce a protectiveresponse against enterotoxigenic E. coli infections. Such protectiondoes not necessarily depend upon bactericidal immune activity. Rather,the protection may result from intestinal secretory IgA antibodiesdirected against enterotoxigenic E. coli colonization factors to preventsmall bowel colonization.

One candidate for a human enterotoxigenic E. coli vaccine is a mixtureof killed wild-type enterotoxigenic E. coli strains chosen for theirexpression of the most common CFAs, supplemented with the recombinant Bsubunit of cholera toxin (chosen as a homolog of the B subunit of LT).It has shown modest efficacy in protecting travelers from diarrhealepisodes, but, although immunogenic, it showed no efficacy againstvaccine preventable events in a large field trial in Egyptian children.

Another candidate for a human enterotoxigenic E. coli vaccine includesenterotoxigenic E. coli CFAs encapsulated in biodegradable,biocompatible poly-lactide-co-glycolide (PLGA) microspheres. Themicrospheres are designed to target M cell uptake with slow release ofthe antigens in mucosal inductive sites. Microsphere vaccines have beentested in human volunteer challenge studies and in an intranasal mousechallenge model. These microsphere vaccines have not been demonstratedto induce consistent immune responses.

A spontaneously derived nontoxigenic LT- and ST-ETEC strain expressingthe CS1 and CS3 components of CFA/II can induce serum and/or intestinalantibody responses to CFA/II components in volunteers vaccinated withthe strain. Moreover, 75% of the volunteers were protected againstchallenge with a wild-type CFA/II ETEC strain of different serotype.Thus, protection against ETEC challenge can be induced by immunity toCFAs. However, this ETEC strain caused some vaccines to develop crampsand diarrhea. Subsequently, this strain has been further attenuated bychromosomal deletion of genes for utilization of aromatic amino acids(aroC) and for outer membrane proteins (e.g., ompR (PTL002) or ompC andompF (PTL003)), which were better tolerated. Despite being welltolerated, development and testing of these attenuated vaccines has beensuspended.

Pathogenic but noninvasive E. coli such as, for example,attaching/effacing E. coli (AEEC) have strong potential as vectors fororal vaccines. Advantages of using AEEC strains for expressing ETECantigens include, for example, maintenance of their native configurationand the targeting of Peyer's patches. We have attenuatedattaching/effacing E. coli strains by modifying virulence genes encodedon the locus of enterocyte effacement (LEE) pathogenicity island (PAI)and have shown these constructs to be safe and effective vaccines. Wenow describe using such strains as vectors to deliver ETEC antigens.

The genes sufficient for the development of Attaching/Effacing (A/E)adherence by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E.coli are contained in a pathogenicity island termed the locus ofenterocyte effacement (LEE). To develop attenuated A/E strains asvaccines and vectors in animal models of intestinal disease we utilizedrabbit enteropathogenic E. coli (REPEC) strains. The LEE of REPEC strainRDEC-1 (O15:H—) was sequenced and found to be highly homologous to theLEEs of EPEC and EHEC. All three LEEs contain a shared core region of 40open reading frames. Among the shared genes, high homology (>95%identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predictedamino acid level was observed for the components of the type IIIsecretion apparatus, and the Ler (LEE-encoded regulator). Moredivergence (66% to 88% identity) was observed in genes encoding proteinsinvolved in host interaction, such as the adhesin intimin (Eae) and thesecreted protein Tir (translocated intimin receptor). We used thissequence information to attenuate REPEC strains by creating in-framedeletion mutants or gene truncations in key virulence genes of the LEE.We then tested these mutants for attenuation, for immunogenicity, andfor protective efficacy against challenge with virulent A/E pathogens.

We have developed a vaccine vector relevant to human disease based on aneae truncation mutant of an O157:H7 shiga toxin producing E. coli (STEC)strain that had been negative for shiga toxin 1 and was deleted forshiga toxin 2. The resulting vector strain is designated herein asZCR533. While ZCR533 has been developed for use as a vaccine vector incattle, O157 EHEC are non-pathogenic in cattle. (U.S. Patent ApplicationPublication No. 2008/0286310 A1). In contrast, O157 EHEC are pathogenicin, for example, mice and humans. Thus, it was unclear whetherattenuation of the vector would necessarily result is a vector-basedvaccine that would be well tolerated in, for example, mice and humans.Here we show that in mice, the O157:H7 Stx1-, Stx2-, Δ-eae strain ZCR533did not produce A/E lesions in vitro, but induced mucosal and serumantibody to intimin. Thus, the strain can serve as a vaccine for humansubjects against O157 EHEC infection.

Construction of the ZCR533 vector is described in U.S. PatentApplication Publication No. 2008/0286310 A1. The vector may be acomponent of a vaccine useful for delivering to a subject one or moreimmunogens whose coding sequence is cloned into and expressed by thevector. Coding sequences for immunogens may be cloned into the vectorusing standard techniques well-known to those skilled in the art.

Any suitable immunogen may be delivered to a subject in this way.Suitable immunogens include, for example, immunogens naturally expressedby enterotoxigenic E. coli such as, for example, heat stabileenterotoxin (ST), heat-labile enterotoxin (LT), colonization factorantigen I (CFA/I), intimin, and lipopolysaccharide (LPS) such as, forexample, LPS naturally expressed by O157 E. coli.

In other cases, however, the vector may be useful more generally todeliver an immunogen to a subject that is naturally expressed by othermicrobes. Thus, exemplary immunogens can include immunogens naturallyexpressed—or derived from immunogens naturally expressed—by, forexample, Gram-negative microbes, Gram-positive microbes, fungi, viruses,and the like.

Thus an immunogen may be expressed by or derived from a protein orpolypeptide expressed by—a Gram-negative microbe. Suitable Gram-negativemicrobes from which an immunogen may be expressed or derives caninclude, for example, an enteropathogen such as, for example, a memberof the family Enterobacteriaceae. Exemplary enteropathogens includemembers of the family Enterobacteriaceae, members of the familyVibrionaceae (including, for instance, Vibrio cholerae), andCampylobacter spp. (including, for instance, C. jejuni). Exemplarymembers of the family Enterobacteriaceae include, for instance, E. coli,Shigella spp., Salmonella spp., Proteus spp., Klebsiella spp. (forinstance, Klebsiella pneumoniae), Serratia spp., and Yersinia spp.Exemplary Salmonella spp. include, for example, Salmonella entericaserovars, Bredeney, Dublin, Agona, Blockley, Enteriditis, Typhimurium,Hadar, Heidelberg, Montevideo, Muenster, Newport senftenberg, Salmonellacholerasuis, and S. typhi. Exemplary strains of E. coli include, forexample, E. coli serotypes O1a, O2a, O78, and O157, different O:Hserotypes including 0104, 0111, 026, 0113, 091, and hemolytic strains ofenterotoxigenic E. coli such as K88⁺, F4⁺, F18ab⁺, and F18ac⁺. Otherexemplary Gram-negative microbes include members of the familyPasteurellaceae (e.g., Pasturella spp. such as, for example, Pasturellamultocida and Pasteurella haemolytica) and members of the familyPseudomonadaceae (e.g., Pseudomonas spp. such as, for example,Pseudomonas aeruginosa). Yet other exemplary Gram-negative microbesinclude, for example, Actinobacillus spp., Haemophilus spp.,Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophagaspp., Flexibacter spp., Flavobacterium spp., Aeromonas spp., and thelike.

In other embodiments, an immunogen may be expressed by—or derived from aprotein or polypeptide expressed by—a Gram-positive microbe. SuitableGram-positive microbes from which an immunogen may be expressed orderived include, for example, members of the family Micrococcaceae suchas, for example, Staphylococcus spp. (e.g., Staphylococcus aureus).Other Gram-positive microbes include members of the familyDeinococcaceae, (e.g., Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, Streptococcus zooepidemicus, orStreptococcus dysgalatiae), Bacillus spp., Corynebacterium spp.,Erysipelothrix spp., Listeria spp., and Mycobacterium spp.,Erysipelothrix spp., and Clostridium spp.

In other embodiments, an immunogen may be expressed by or derived from aprotein or polypeptide expressed by a fungus. Suitable fungi from whichan immunogen may be expressed or derived include, for example,Cryptococcus spp., Blastomyces spp. (e.g., B. deratitidis), Histoplasmaspp. Coccidiodes spp., Candida spp. (e.g., C. albicans), and Aspergillusspp.

In other embodiments, an immunogen may be—or be derived from—a viralprotein or polypeptide. Suitable viruses from which an immunogen may beidentified or derived include, for example DNA viruses and RNA virusessuch as, for example, picornaviruses (e.g., enteroviruses, rhinoviruses,aphthoviruses, and hepatoviruses), coronaviruses, flaviviruses,hepaciviruses, morbilliviruses, metapneumoviruses, rubulaviruses, andlentiviruses.

In another aspect, the present invention provides a vector that caninduce an immune response against both one or more native EHECimmunogens and one or more heterologous immunogens cloned into andexpressed by the vector. In this aspect, the invention provides a toolby which vaccination against multiple pathogens (e.g., EHEC and ETEC,EHEC and Brucella spp.) can be achieved. It is not a given thatimmunogenicity and/or protection versus EHEC challenge can be maintainedwhen the vector expresses one or more heterologous immunogens because itis possible, for example, that expression of one or more heterologousimmunogens can interfere with and/or overwhelm an immune responseagainst EHEC immunogens. Table 3 shows that mucosal antibody responsesto exemplary EHEC immunogens intimin and O157 LPS were maintained whenthe vector was modified to express ETEC immunogens.

As used herein a subject can be an animal such as, for example, humans,dogs, cats, cattle, horses, sheep, swine, rodents, and the like. In somecases, the subject animal can be livestock (e.g., cattle, horses, swine,etc.) or a companion animal (e.g., dogs, cats, etc.). In some cases, thesubject animal is an animal in which wild-type EHEC is a pathogen.

In another aspect, the invention includes a small animal model for thestudy of immunogenicity and efficacy of the vaccines described hereinfollowing ETEC challenge.

Lack of a small animal model has hampered the study of pathogenesis andimmunogenicity of ETEC infection. Some studies involving ETEC haveutilized the suckling mouse for ST effects, rats, guinea pigs, orrabbits for LT effects. However, the species-specificity of ETECadhesins for receptors present in human intestine has prevented theestablishment of an animal model for small bowel adherence andcolonization with human isolates. Transient obstruction of the smallintestine with a removable intestinal tie in an adult rabbit diarrheamodel (RITARD) has permitted small bowel colonization to occur withoutadherence, but this procedure is surgically invasive and does not permitthe study of the contribution of CFAs to pathogenesis.

Intranasal challenge models using mice for the study of theimmunopathogenesis of an enteric infection with Shigella, anotherhost-restricted pathogen, have exploited the mouse lung as a simplifiedmodel for the study of the pathogenesis and immunobiology of Shigellainfection. The mucosal surface of the bronchus of the mouse shares somecharacteristics with the intestinal epithelium mucosal surface, althoughthe tracheobronchi of the mouse have ciliated columnar epithelium andthe alveolar sacs cuboidal epithelium. The bronchial wall containslymphoid follicles that are similar to mouse intestinal Peyer's patchesand the lungs having antigen-presenting cells as well as B lymphocytesand T helper/suppressor lymphocytes. In addition, systemic and localimmune responses can be accurately evaluated after intranasal challengesince the mouse lung is immunologically naive with respect to specificantigens. Moreover, the absence of commensal bacteria in the bronchiallumen means that the lung has not been primed by previous exposure toother Gram-negative bacteria. The intranasal administration of bothShigella spp. and Campylobacter jejuni to mice has been used to studyvaccine candidates, pathogenesis, and vaccination-acquired immunity.

The utility of an intranasal challenge model is supported by theacceptance of the intranasal route as an effective means of stimulatingmucosal immunity, which is sparing of antigen and which results inincreased IgA responses at mucosal surfaces within the common mucosalimmune system. This is the desired type of immunity to protect againstnon-invasive pathogens, like ETEC, which colonize mucosal surfaces.Furthermore, protection can be achieved at distant mucosal sites (suchas the intestine) via intranasal vaccination. Moreover, intranasalimmunization can also induce systemic immune responses. Nasal-associatedlymphoid tissue (HALT) of intranasally immunized mice, but not of naïvemice, contains antigen-specific IgA antibody secreting cells (ASC),indicating that B-cell activation and IgA isotype switching occurs inNALT. NALT serves as an inductive site for antigen-specific IgA ASC thatmigrate to distant mucosal effector sites following intranasalimmunization. Furthermore, memory T cells reside in NALT for extendedperiods. The presence of NALT in the upper respiratory tract and theability to disseminate antigen-specific IgA ASC to distant mucosaleffector sites support use of intranasal immunization.

We describe herein an intranasal challenge model using the ETEC strainsH10407 and B7A, and use this model to effectively study theimmunogenicity and efficacy of CFA and toxin-based vaccines. Althoughphysiologic conditions present in the GI tract, but absent in therespiratory tract (including acidic pH, proteolytic enzymes, bile salts,peristalsis, the microbiota, and usual target cells for bacterial andtoxin binding), may affect the course of an ETEC infection and theexpression of ETEC antigens, the intranasal mouse model can neverthelesspermit the valid study of anti-CFA and anti-toxin immunity followingimmunization and challenge. Thus, the intranasal route of inoculationcan be used effectively both for mucosal immunization with ETEC antigensand to produce a reproducible model of ETEC infection resulting inmultifocal bronchopneumonia with no evidence of systemic spread outsidethe lungs.

Vaccine candidates containing plasmids expressing either CFA/I, alone,or CFA/I plus mutant E. coli LT express CFA/I fimbriae on their surface.CFA/I fimbriae manifest both hemagglutinating and hydrophobic propertiescharacteristic of the wild-type fimbriae. In addition, the vaccine withthe mLT encoding plasmids expressed LT in its periplasmic space. Both ofthe expression plasmids tested proved to be stable in 70% of bacteriaafter 100 generations in vitro and were stable in vivo for 10 days afterintranasal administration. A summary of the properties of the vectors isprovided in FIG. 1

To establish that our ZCR533 EHEC vector and its related vaccine strainscan be safely administered intranasally to mice at doses which areimmunogenic, we administered a range of doses (from 1×10⁷ to 5×10⁹) ofthe ZCR533 EHEC vector and of the EHEC(CFA/I) and EHEC (CFA/I-mLT)vaccines strains intranasally to mice in a volume of 20 μl.

Safety was determined by clinical observation of signs of distress.Doses of up 5×10⁷ bacteria of the vaccine vector (or its derivativeexpressing CFA/I and mLT) administered intranasally in a 20 μl volumecaused no distress in any animals. Animals receiving up to 1×10⁹ showedonly mild and transient distress.

Immunogenicity for a single dose (or two doses) was assessed by: (a) theability of sera obtained at 14 days after the final dose to agglutinatea suspension of the vector ZCR533 and the wild-type (WT) H10407, and (b)ELISAs against whole cells of the EHEC ZCR533 vector and of WT H10407expressing CFA/I. Results are shown in Table 1.

TABLE 1 Serum Antibody Responses Induced by Intranasal ImmunizationsSerum against ZCR533 Serum against ZCR533 Serum against ETEC (CFA/I-mLT)Vaccine EHEC Vector Strain H10407 WT Strain Strain Slide Bacterialagglutination Positive 2 doses (5 × 10⁷ Negative Positive 2 doses (5 ×10⁷ of ZCR533 vector CFU) CFU) Slide Bacterial Agglutination Negative (1× 10⁷ to Positive (1 × 10⁷ to Positive (1 × 10⁷ to 5 × 10⁹ of WT H104075 × 10⁹ CFU) 1 × 10⁹ CFU) CFU) Serum ELISA IgG titers +1/60 at (5 × 10⁷CFU) Negative (1 × 10⁷ to +1/60 at (1 × 10⁸ CFU) against ZCR533 vector+1/180 at (1 × 10⁸ CFU) 5 × 10⁹ CFU) +1/180 at (5 × 10⁸ CFU) Serum ELISAIgG titers Negative (1 × 10⁷ to +1/180 at (1 × 10⁷ +1/180 at (1 × 10⁷CFU) against whole cell H10407 5 × 10⁹ CFU) CFU) +1/540 at +1/540 at (5× 10⁸ CFU) (1 × 10⁹ CFU) +1/1620 at (5 × 10⁹ CFU)

These results indicate that an immune response against the vector can beinduced by two immunizations with a safe dose (5×10⁷ CFU) of the vaccinevector itself. Similar safe single doses of the ZCR533 (CFA/I-mLT)vaccine strain induced slide agglutination of the WT CFA/I+H10407 aswell as measurable ELISA titers to these organisms. The ELISA titers andagglutinating activity induced by the vaccine was comparable to thoseinduced by intranasal administration of the WT H10407 at sub lethaldoses.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

Female BALB/c mice (6-8 weeks of age) were anesthetized with isofluraneand a range of doses (1×10⁷-1×10⁸ CFUs) of the vaccine construct(ZCR533-CFA/I and ZCR533-CFA/I plus mLT) administered in a 20 μl volumedrop-wise to the external nares of each mouse. Following intranasaldelivery of the vaccine constructs, the mice were observed for adverseeffects for at least 14 days following administration of the vaccineconstructs. The mice were monitored for adverse effects includingruffled fur, huddling, lethargy, shivering, labored breathing,difficulty in moving, refusal to eat or drink, drainage from eyes,hunched posture and/or moribund condition. Results are shown in Table 2.

TABLE 2 Effect of vector and vaccine constructs on BALB/c mice followingintranasal delivery Strain 1 × 10⁷ CFU 5 × 10⁷ CFU 1 × 10⁸ CFU ZCR533 NN N ZCR533-CFA/I N N N ZCR533-CFA/I + N N N mLT N = No significant signsof distress in the mice

Example 2

Mice were administered intranasally two doses of the vaccine constructs(5×10⁷ bacteria per dose) in a 20 μl volume with an interval of 14 days.Fourteen days following the second dose of vaccine constructs, blood andmucosal collections (fecal pellets, nasal and lung lavages, and smalland large intestinal washes) were obtained. Blood was collected from themice by tail nick with a razor blade, clotted overnight at 4° C.,centrifuged at 10,000×g for 10 minutes and the sera stored at −80° C.Fecal pellets were obtained by placing mice on absorbent paper under a600-ml beaker for 10-15 minutes. Fecal pellets were placed in microfugetubes with 1 ml of Protease Inhibitor Cocktail (PIC) (Sigma Chemical Co.St. Louis, Mo.), broken up with sterile toothpicks, centrifuged at10,000×g for 15 minutes and supernatants stored at −80° C.

Lung lavages, nasal lavages, and small intestinal washes, and largeintestinal washes were collected from the mice following euthanasiausing carbon dioxide. For lung lavages, a catheter was inserted into thetrachea and 1 ml of PIC used to inflate the lungs. For nasal lavages, acatheter was inserted into the trachea and 500 μl of PIC used to flushthe nasal area with the fluid being collected from the nares. For smallintestinal washes, the small intestines were cut about 1-2 cm from thestomach and about 1-2 cm from the cecum, and 2 ml of PIC flushed throughthe small intestines. For large intestinal washes, the large intestineswere cut at the cecum and at the anus, and 500 μl of PIC flushed throughthe large intestines. All washes and lavages were centrifuged at10,000×g for 15 minutes and the supernatants stored at −80° C.

Serum (IgG, IgA, and IgM) and mucosal (IgA and IgG) antibodies againstCFA/I, LT, intimin and O157 LPS were measured by the use of an ELISA(Byrd, W. and F. J. Cassels. 2006. Microbiology 152:779-786) afteradministration of either (1) ZCR533, (2) ZCR533-CFA/I, or (3)ZCR533-CFA/I+mLT construct. The mucosal sample concentrations weredetermined from standard curves using purified mouse IgA and IgGantibody, and the CFA/I or LT-specific antibody concentrations werenormalized based on total IgA and IgG content, with values expressed asμg CFA/I or LT-specific IgA or IgG/mg total IgA or IgG. Results areshown in FIGS. 2-29.

Example 3 CFA/I Binding Inhibition Assay

ETEC wild-type H10407 possessing CFA/I were used to assay for bindinginhibition to Caco-2 cells. The Caco-2 cells were grown to confluence ina culture flask, trypsinized and distributed onto sterile chamber glassslides. ETEC bacteria (1×10⁹) were incubated with mouse serum raisedagainst the ZCR533 vector, and the ZCR533-CFA/I and ZCR533-CFA/I plusmLT vaccine constructs, for 30 minutes. Anti-CFA/I and non-immune mouseserums were used as positive and negative controls, respectively. Thebacterial mixture was added to the Caco-2 cells and incubated for 3hours at 37° C. The Caco-2 cells were washed with PBS and fixed with 70%methanol for 10 minutes, and then the fixed cells were stained with 20%Giemsa stain (Sigma). The slide containing the Caco-2 cells and the ETECH10407 bacteria were examined under a microscope at 400×-1,000×magnification and the percentage of Caco-2 cells with at least oneadherent ETEC bacterial cell was determined. The results were averagedfor three separate fields each with 50 Caco-2 cells per field. Resultsare shown in FIG. 30.

Example 4 LT Inhibition Assay

CHO Cell Elongation

LT inhibition was determined by measuring extent of Chinese hamsterovary (CHO-K1) cell elongation in the presence of LT essentially aspreviously described (Ranallo, R. T., et al., 2005. Infect. Immun.73:258-267). LT (List) (25 ng) was incubated with mouse serum raisedagainst the ZCR533 vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct, for 30 minutes. Toxin-antiserummixtures were added to trypsinized CHO cells and assayed for elongation.Anti-LT and non-immune mouse serums were used as positive and negativecontrols, respectively. The results were averaged for three separatefields each with 100 CHO cells per field. CHO elongation with no LT wasaround 15% of the cells and with 25 ng LT around 90% of the cells.Results are shown in FIG. 31.

GM1 Inhibition Assay

LT inhibition was also determined by measuring extent of inhibition ofLT and the B subunit of LT (LT-B) from binding to its GM1 receptor thepresence of LT essentially as previously described (Moravec, T., 2006.Vaccine 25:1647-1657). LT (List) or LT-B (Sigma) were incubated withmouse serum raised against the ZCR533 vector, the ZCR533-CFA/I vaccineconstruct, or the ZCR533-CFA/I+mLT vaccine construct, for 30 minutes.Toxin-antiserum mixtures were added to wells of ELISA plates coated withGM1 and a standard ELISA performed. Anti-LT and non-immune mouse serumswere used as positive and negative controls, respectively. Results areshown in FIG. 32 and FIG. 33.

Example 5 Active Immunization

Mice were vaccinated with two doses (5×10⁷ CFU/20 μl each dose) of theZCR533 vector, ZCR533-CFA/I vaccine construct, or the ZCR533-CFA/I+mLTvaccine construct. The second dose was administered 14 days after theinitial dose.

The vaccinated mice were intranasally challenged with a lethal dose ofwild-type ETEC strain H10407 (3×10⁹ bacteria) in a volume of 25 μl 15days following second vaccination. The mice were examined for mortalityand morbidity at least twice daily for 14 days post challenge. Theclinical signs of distress monitored were weight loss, ruffled fur,huddling, lethargy, shivering, labored breathing, difficulty in moving,refusal to eat or drink, diarrhea, drainage from eyes, shivering,hunched posture and moribund condition. Results are shown in Table 3.

TABLE 3 Active immunization ZCR533 CFA/I CFA/I + mLT Vector VaccineVaccine Days PBS Diluent Strain Strain Strain 0 20/20 Alive 20/20 Alive20/20 Alive 20/20 Alive 1 20/20 Alive 20/20 Alive 20/20 Alive 20/20Alive 2 12/20 Alive 12/20 Alive 17/20 Alive 18/20 Alive 3 0/20 Alive0/20 Alive 13/20 Alive 13/20 Alive 4 0/20 Alive 0/20 Alive 12/20 Alive11/20 Alive 5 0/20 Alive 0/20 Alive 12/20 Alive 11/20 Alive % 0/20 0/2012/20 11/20 total Survival 0% Survival 0% Survival 60% Survival 55%Survival (P < 0.05 vs. (P < 0.05 vs. sham mice) sham mice)

Example 6 Passive Immunization

Mice were intranasally challenged with a lethal dose of wild-type ETECstrain H10407 (3×10⁹ bacteria/25) previously incubated for 1 hour with a1:10 dilution of serum collected from mice vaccinated with two doses ofeither the ZCR533 vector, the ZCR533-CFA/I vaccine construct, or theZCR533-CFA/I+mLT vaccine construct. The serum was prepared byvaccinating mice with two doses (5×10⁷ bacteria/20 μl, each dose, 14days between doses) of either the ZCR533 vector, the ZCR533-CFA/Ivaccine construct, or the ZCR533-CFA/I+mLT vaccine construct. 15 daysafter the second vaccination, the mice were sacrifice and bled, and theserum collected and prepared using standard methods. The serum wasdiluted ten-fold prior to being incubated with the ETEC strain H10407.

The mice challenged with serum-incubated ETEC strain H10407 wereexamined for mortality and morbidity at least twice daily for 14 dayspost challenge as described above for the assessment of activeimmunization. Results are shown in Table 4.

TABLE 4 Passive Immunization ZCR533 CFA/I + mLT Vector CFA/I VaccineVaccine Days PBS Diluent Strain Strain Strain 0 10/10 Alive 10/10 Alive10/10 Alive 10/10 Alive 1 10/10 Alive 10/10 Alive 10/10 Alive 10/10Alive 2 2/10 Alive 3/10 Alive 8/10 Alive 9/10 Alive 3 0/10 Alive 0/10Alive 6/10 Alive 8/10 Alive 4 0/10 Alive 0/10 Alive 6/10 Alive 6/10Alive 5 0/10 Alive 0/10 Alive 6/10 Alive 6/10 Alive % 0/10 total 0/106/10 6/10 Survival 0% Survival 0% Survival 60% Survival 60% Survival

Example 7 Lung Clearance

Mice were vaccinated twice (5×10⁷ CFU/20 μl per vaccination, 14 daysbetween vaccinations). 15 days after the second vaccination, thevaccinated mice were intranasally challenged with a sublethal dose ofwild-type ETEC strain H10407 (approximately 1×10⁹ bacteria) in a volumeof 25 μl 15 days following immunization with the ZCR vector (ZCR), theZCR533-CFA/I vaccine construct (CFA/I), or the ZCR533-CFA/I+mLT vaccineconstruct (mLT). The mice were euthanized, and the lungs asepticallyremoved and placed in 2 ml of sterile PBS. The lungs were homogenizedusing Potter-Elvehjem glass tissue grinders to free bacteria intosuspension, and the homogenates plated to determine the number ofbacteria present in the lungs at the indicated times (24, 48, 72, 120,and 168 hours) post challenge. Results are shown in FIG. 34.

Example 8

Bacterial colonization of the small intestines and intestinal fluidaccumulation were measured in mice previously vaccinated with theZCR533-CFA/I or ZCR533-CFA/I+mLT vaccine constructs. The ability of thevaccine constructs to induce an immune response in the mice sufficientto significantly reduce ETEC H10407 bacterial colonization of the smallintestines and reduce intestinal fluid accumulation induced by the LTalone or ETEC H10407 bacteria was measured. The mice were eithervaccinated intranasally (as described above) (two doses, 14-day intervalbetween doses, 5×10⁷ bacteria/20 μl per dose) or intragastrically (onedose given daily for three consecutive days, repeated three times with a14-day interval between each three consecutive days dosing, 3×10⁹bacteria/200 μl per dose). Each mouse was intragastrically administeredusing a 20-gauge ball-tip needle 100 μl sterile 10% sodium bicarbonate.Thirty minutes later a suspension of either vaccine construct (3×10⁹bacteria) in a volume of 200 μl was intragastrically administered to themice.

Bacterial Colonization.

Vaccinated mice received sterile drinking water containing streptomycin(5 g/liter) 72 hours prior to ETEC H10407 intragastric administration toeradicate normal resident flora in the intestinal tract. Thestreptomycin-treated water contained 5% fructose to encourage waterconsumption. Streptomycin-treated water was replaced with sterile normalwater (no streptomycin) 6 hours prior to ETEC H10407 inoculation. Eachmouse was intragastrically administered using a 20-gauge ball-tip needle100 μl sterile 10% sodium bicarbonate. Thirty minutes later a suspensionof ETEC H10407 (3×10⁹ bacteria) in a volume of 300 tad wasintragastrically administered to the mice. Mice were euthanized 24 hoursfollowing ETEC H10407 inoculation and ETEC H10407 bacteria harvestedfrom the small intestines. The small intestines were aseptically removedand placed into 2 ml of sterile 5% saponin solution, vortexed for 5-10seconds, incubated for 10 minutes at room temperature and vortexed asecond time for 5-10 seconds. The small intestines were homogenizedusing Potter-Elvehjem glass tissue grinders to free bacteria intosuspension. The resulting suspensions were plated to determine thenumber of bacteria present in the small intestines of each mouse.Results after intranasal vaccination are shown in FIG. 35. Results afterintragastric vaccination are shown in FIG. 36.

Fluid Accumulation (LT)

Vaccinated mice were intragastrically administered using a 20-gaugeball-tip needle 100 μl sterile 10% sodium bicarbonate. Thirty minuteslater a suspension of LT (25 μg) in a volume of 400 μl wasintragastrically administered to the mice. Mice were euthanized 3 hoursfollowing LT administration and the entire intestines (from duodenum toanus) were carefully removed to retain any accumulated fluid. Fluidaccumulation was determined by weighing the intestines. The carcass wasweighted separately and a gut/carcass ratio was determined for eachmouse. Results are shown in FIG. 37.

Fluid Accumulation (ETEC)

Vaccinated mice were intragastrically administered using a 20-gaugeball-tip needle 100 μl sterile 10% sodium bicarbonate. Thirty minuteslater a suspension of ETEC H10407 (3×10⁹ bacteria) in a volume of 300 μlintragastrically administered to the mice. Mice were euthanized 3 hoursfollowing ETEC H10407 administration and the entire intestines (fromduodenum to anus) were carefully removed to retain any accumulatedfluid. Fluid accumulation was determined by weighing the intestine's.The carcass was weighted separately and a gut/carcass ratio wasdetermined for each mouse. Results are shown in FIG. 38.

Example 9

Mice are vaccinated with two doses (5×10⁷ CFU/20 μl each dose) of theZCR533 vector. The second dose was administered 14 days after theinitial dose. 15 days after the second vaccination, the vaccinated micewere intranasally challenged with a sublethal dose of wild-type EHECstrain (approximately 1×10⁹ bacteria).

Active immunization is assessed as described in Example 5. Micevaccinated with the ZRC533 vector will exhibit improved protectionagainst wild-type EHEC challenge compared to the negative control.

Passive immunization is assessed as described in Example 6. Micechallenged with a lethal dose of wild-type EHEC strain (3×10⁹bacteria/25 μl) previously incubated for 1 hour with a 1:10 dilution ofserum collected from mice vaccinated with two doses of the ZCR533 vectorwill exhibit improved protection compared to the negative control.

Lung Clearance is assessed as described in Example 7. The lungs of micevaccinated with the ZCR533 vector will have fewer EHEC bacteria thanunvaccinated mice.

Bacterial colonization is assessed as described in Example 8. The smallintestines of mice vaccinated with the ZCR533 vector will exhibitreduced EHEC colonization compared to unvaccinated mice.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method of generating an antibody response in amammalian subject against a mutant heat-labile toxin (LT) of anenterotoxigenic E. coli (ETEC) and a heterologous microbial immunogen,the method comprising administration to said subject, a compositioncomprising an attenuated enterohemorrhagic E. coli (EHEC) in an amounteffective to induce the antibody response, wherein the attenuated EHECis modified to comprise: (a) truncation of the intimin adhesin by amutation of the coding region of its eae gene at the locus of enterocyteeffacement (LEE); (b) a plasmid expressing the mutant LT, and (c) aplasmid expressing the heterologous microbial antigen, wherein themutant LT is expressed in the periplasmic space.
 2. The method of claim1, wherein the administration is intranasal administration.
 3. Themethod of claim 1, wherein the heterologous microbial immunogen is thecolonization factor antigen I (CFA/I) of an enterotoxigenic E. coli(ETEC).
 4. The method of claim 2, wherein the antibody responsegenerated in the subject is a serum antibody response and a mucosalantibody response against a wild-type ETEC and a wild-type EHEC.
 5. Themethod of claim 4, wherein the antibody response protects the subjectagainst a challenge infection by a wild-type ETEC.
 6. The method ofclaim 4, wherein the antibody response protects the subject against achallenge infection by a wild-type EHEC.